The present invention relates generally to cytokine inhibitors. More particularly, the invention relates to inhibitors to IL8 receptor binding, including antibodies, that interact with the amino-terminal extracellular domain of the IL8 receptor and compete with IL8 and other related natural ligands, for receptor-binding.
Cytokines are a group of hormone-like mediators produced by leukocytes. These agents serve as endogenous biological signals that act in conjunction with antigens to amplify both localized and systemic host defense mechanisms involving macrophages, lymphocytes, and other cell types. Representative cytokines include the various interleukins, interferons, GROxcex1, GROxcex2 and GROxcex3, neutrophil activating peptide-2 (NAP-2) and ENA-78. Cytokines have been used to treat and prevent a wide variety of disorders based on the ability of these molecules to stimulate an immunological response.
Interleukin-8 (IL8) is a cytokine originally derived from human macrophages (Suzuki, K. et al. (1989) J. Exp. Med. 169:1895-1901; Schrxc3x6der, J. M. et al. (1987) J. Immunol. 139:3474-3483; Schrxc3x6der, J. M. et al. (1988) J. Immunol. 140:3534-3540; Schrxc3x6der, J. M. (1989) J. Exp. Med. 170:847-861; Larson, C. G. et al. (1989) Science 243:1464-1466). This factor has also been called polymorphonuclear (PMN) chemotactic factor, monocyte-derived neutrophil-activating peptide (MONAP), monocyte-derived neutrophil chemotactic factor (MDNCF), T lymphocyte chemotactic factor (TCF), lymphocyte-derived neutrophil-activating peptide (LYNAP), and neutrophil-activating peptide 1 (NAP-1).
The IL8 molecule is produced by a wide variety of tissues and cells, including mononuclear phagocytes, endothelial cells, fibroblasts, epithelial cells and alveolar macrophages, upon stimulation with such agents as lipopolysaccharide and phorbol myristate, phytohemagglutinin, Con A, or other mitogenic preparations and cytokines such as interleukin-1 (IL1) and tumor necrosis factor (TNF). The gene coding for human IL8 has been cloned. Matsushima, K. et al. J. Exp. Med. (1988) 167:1883-1893; Mukaida, N. et al. J. Immunol. (1989) 143:1366-1371. The gene encodes a precursor protein having 99 amino acids which is proteolytically cleaved into secreted IL8 polypeptides of various lengths, such as those containing 69, 72 or 77 amino acid residues, with molecular masses of approximately 8000 Daltons.
Human IL8 acts as a chemoattractant for neutrophils, and induces granulocytosis upon systemic injection and skin reaction upon local injection, in experimental animals. Bazzoni, F. et al. (1991) 173:771-774; Van Damme, J. et al. (1988) J. Exp. Med. 167:1364-1376; Ribeiro, R. A. et al. (1991) Immunology 73:472-477. The molecule also activates the release of superoxide anions and elicits release of the primary granule constituents of neutrophils, including myeloperoxidase, xcex2-glucuronidase and elastase. IL8 mediates these activities by binding to its receptor and triggering signal transduction, a cascade of reactions ultimately resulting in a biological response.
The sequences of two human IL8 receptors (termed xe2x80x9cIL8R1xe2x80x9d and xe2x80x9cIL8R2xe2x80x9d herein), have been reported. See, e.g., International Publication No. WO93/06229 (published Apr. 1, 1993) and Holmes et al. Science (1991) 253:1278-1280. These receptors have a similar affinity for IL8 and are members of the rhodopsin seven-helix membrane-spanning superfamily. These receptor molecules include seven transmembrane regions, linked by three intracellular and three extracellular loops and possess an extracellular amino-terminal tail and an intracellular carboxy-terminal tail. Other naturally occurring cytokines are known to share a receptor with IL8. For example, GROxcex1, GROxcex2, GROxcex3, NAP-2 and ENA-78, all bind to one IL8 receptor on human neutrophils, as described in Baggiolini et al., FEBS Lett. (1992) 307:97-101; Walz et al., J. Exp. Med. (1991) 174:1355; Moser et al., J. Biol. Chem. (1991) 266:10666; Geiser et al., J. Biol. Chem. (1993) 268:15419-15424.
Gayle et al., J. Biol. Chem. (1993) 268:7283-7289 and LaRosa et al., J. Biol. Chem. (1992) 267:25402-25406 relate to an important determinant for the binding of natural IL8R2 agonists, GROxcex1/MGSA and NAP-2, resides in the region of the receptor that includes the amino-terminal extracellular domain and a portion of the first transmembrahe region.
Peptides based on the sequence of the IL8 receptor have been constructed in an effort to determine the role of the various regions of the IL8 receptor in IL8 binding. Gayle et al., J. Biol. Chem. (1993) 268:7283-7289 describes peptides based on the amino-terminal sequence of the IL8 receptor and International Publication No. WO92/04372 (published Mar. 19, 1992) describes peptides and antibodies thereto based on the carboxy terminus of the receptor.
The present invention provides for substances that inhibit IL8 binding to its receptor via interaction with the amino-terminal extracellular domain of the IL8 receptor. The inhibitors are useful modulators of IL8 receptor-mediated biological activity.
Accordingly, in one embodiment, the invention is directed to an inhibitor to IL8 receptor-binding, as defined below, that comprises a molecule that is capable of interacting with the amino-terminal extracellular domain of an IL8 receptor and that is capable of competing with IL8 for the receptor.
In a preferred embodiment, the inhibitor is an antibody raised against a polypeptide derived from the amino-terminal extracellular domain of IL8R1 or IL8R2 comprising an amino acid sequence substantially the same as any one of the amino acid sequences M-S-N-I-T-D-P-Q-M-W-D-F-D-D-L, (SEQ ID NO:1) M-E-S-D-S-F-E-D-F-W-K-G-E-D-L, (SEQ ID NO:2) F-E-D-F-W-K-G-E-D-L-S-N-Y-S-Y, S-S-T-L-P-P-F-L-L-D-A-A-P-C (SEQ ID NO:3) or F-L-L-D-A-A-P-C-E-P-E-S-L-E-I (SEQ ID NO:5).
In another preferred embodiment, the invention includes a mixture of antibodies raised against at least four polypeptides derived from the amino-terminal extracellular domain of IL8R2, wherein
(i) the amino acid sequence of the first polypeptide comprises an amino acid sequence substantially the same as the amino acid sequence M-E-S-D-S-F-E-D-F-W-K-G-E-D-I (SEQ ID NO:2);
(ii) the amino acid sequence of the second polypeptide comprises an amino acid sequence substantially the same as the amino acid sequence F-E-D-F-W-K-G-E-D-L-S-N-Y-S-Y (SEQ ID NO:3);
(iii) the amino acid sequence of the third polypeptide comprises an amino acid sequence substantially the same as the amino acid sequence S-S-T-L-P-P-F-L-L-D-A-A-P-C (SEQ ID NO:4); and
(iv) the amino acid sequence of the fourth polypeptide comprises an amino acid sequence substantially the same as the amino acid sequence F-L-L-D-A-A-P-C-E-P-E-S-L-E-I (SEQ ID NO:5).
In another embodiment, the invention is directed to a method of inhibiting the binding of IL8 to its receptor, comprising:
(a) providing an inhibitor to IL8 receptor binding that is capable of binding to the amino-terminal extracellular domain of an IL8 receptor and is also capable of competing with IL8 for the receptor; and
(b) contacting the receptor with an effective inhibiting amount of the inhibitor.
In yet another embodiment, the invention is directed to a method of modulating an IL8 receptor-mediated biological response comprising:
(a) providing an inhibitor to IL8 receptor binding that is capable of binding to the amino-terminal extracellular domain of an IL8 receptor and that is capable of competing with IL8 for the receptor; and
(b) contacting a cell that produces an IL8 receptor with an effective modulating amount of the inhibitor.
In another embodiment, the invention is directed to an inhibitor to IL8 receptor-binding comprising a molecule that is capable of interacting with the amino-terminal extracellular domain of an IL8 receptor and that is capable of competing with a molecule selected from the group consisting of IL8, GROxcex1, GROxcex2, GROxcex3, NAP-2 and ENA-78, for the receptor.
These and other embodiments of the subject invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.